Breast cancer is the most common malignancy in women in North America. Each year more than 210,000 new cases of breast cancer are diagnosed in the US (1-3). In the clinic, endocrine therapy is an important intervention for cancers that express estrogen receptor (ER), and it has proven to be one of the most effective treatment strategies for breast cancer (3,4). At diagnosis, about 70% of breast cancers contain estrogen receptors and depend on estrogen for growth and progression. Expression of ER in a tumor is predictive of a clinical response to hormonal therapy. Such observations have led to current use of antiestrogens (such as fulvestrant, tamoxifen and its relatives, raloxifene, toremifene, lasofoxifene, etc.) and aromatase inhibitors in treating ER-positive breast cancer (2,3). Tamoxifen and its analogues are among the most highly prescribed drugs for initial estrogen-dependent breast cancer. However, they are not without their drawbacks since they bind to the estrogen receptor in many tissues (bone, uterus, etc.) and can have harmful effects. A substantial proportion of patients presenting with localized disease, and all of the patients with metastatic breast cancer, become resistant to current endocrine therapies (5, 6). Thus, there is an urgent need to develop alternative therapeutics to overcome endocrine resistance and to improve the long-term survival of afflicted patients. Despite remarkable improvements in treatment options, development of endocrine resistance is one reason that breast cancer is the second most frequent cause of cancer death in women (5-7). In most cases, the ER is present in resistant tumors, and in many of these its activity continues to regulate tumor growth.
Classical and nonclassical mechanisms of estrogen action in breast malignancy. Estrogen modulates gene transcription in breast cancers through its receptors using different signaling pathways (2,8) (see FIG. 1). The classical pathway involves direct DNA binding of liganded receptor to estrogen response elements (EREs) in the promoter regions of responsive genes.
The proliferation and survival of breast cancers is closely regulated by growth factor receptors as well as estrogens (E2) and their receptors, estrogen receptor (ER)-α and -β, with ERα generally considered most important in tumor progression (5,6,9). ERα has 6 major functional domains including an N-terminal transactivation domain, an adjacent DNA-binding domain and a C-terminal portion involved in hormone-binding, receptor dimerization and activity of a second transactivation region. In classical models of E2 action, E2 binds ER to promote dimerization and phosphorylation of the receptor. This allows direct binding of the ligand-ER complex with steroid receptor coactivators (CoReg) and E2-responsive elements (ERE) in DNA, leading to changes in gene transcription that regulate growth, differentiation, apoptosis and angiogenesis. In addition, there are alternate pathways of E2 action that involve protein-protein interactions and do not require direct ER binding to DNA. A subset of ER associate with extranuclear sites and interact there with membrane growth factor receptors (EGFR, HER2) and other signaling molecules (components of the ras-MAPK and PI3K/AKT pathways, Shc, src kinases, JAK/STAT, nitric oxide synthase (NOS), G-proteins). Of special note, growth factor and extranuclear estrogen receptors appear to form a structured complex for signal transduction to MAPK and/or PI3K/AKT kinase that interacts, in turn, with nuclear ER and CoReg. Signaling for cell growth involves phosphorylation (P) of nuclear ER and CoReg, and such phosphorylation can occur in ligand-dependent as well as ligand-independent modes. ERE-dependent and alternate transcription sites may be activated. Further, E2 is produced locally in supporting cells by the action of aromatase (ARO), and ARO is regulated by both nulcear and extranuclear ER and growth factor-mediated signaling. In addition, estrogens may regulate tumor-associated angiogenesis by direct interactions with vascular endothelial cells or by indirect stimulation of VEGF secretion from tumors.
However, it is now clear that the ERα can regulate genes that lack a canonical ERE, suggesting additional pathways for estrogen action that may be of paramount importance in modulating tumor progression. Alternate, nonclassical pathways involve indirect modulation of transcription by ER interaction with components of other transcription complexes (AP-1, nuclear factor-kB) or kinase signaling complexes (MAPK, PI3K/AKT kinase) via protein-protein interactions. Emerging data suggest that interactions of ER with growth factor receptor-kinase signaling pathways may play a critical role in promoting estrogen signaling for tumor progression (9). Based on current data in estrogen target cells, nonclassical ER signaling is associated with epithelial proliferation but not other estrogen-responsive events such as fluid accumulation in uterus (8), while classical ER signaling appears more essential for skeletal development, bone health and other differentiated cell functions (10).
ER often continues to play a major role in controlling growth of hormone-resistant cancers. In treatment with aromatase inhibitors (AI's), ER activation by alternate ligands, local E2 production and development of ER hypersensitivity are especially problemsome (2,6). In addition, ligand-independent activation of ER occurs in tumors overexpressing growth factor receptors such as HER2, with growth factor receptors promoting ER phosphorylation even in the absence of estrogen (5,9,11). Such ligand-independent mechanisms likely contribute to resistance to AI's as well as antiestrogens (12,13). These nonclassical events are mediated by ER or adaptor proteins that impact gene expression indirectly by activating growth-promoting kinase cascades to regulate transcription. In breast tumors, significant evidence suggests that regulation of both proliferation and cell death pathways occurs, in part, by the action of nonclassical kinase-mediated pathways (9,11,14-19). Better understanding and targeting of these complex signaling pathways in tumors with endocrine resistance to both antiestrogens and AI's will help in development of individualized and improved treatments in the clinic.
Current antiestrogens are competitive antagonists of estrogen and disrupt ER-induced transcription. However, some antagonists display partial estrogenic activity in a tissue- and gene-dependent manner, hence their description as selective estrogen receptor modulators (SERMs). Tamoxifen, a partial agonist that limits effects of E2 in breast, has been the most widely used hormone therapy for the past 20 years, achieving a 39% reduction in disease recurrence and a 31% reduction in mortality in ER+ early breast cancer (6,20,21). Although effective, tamoxifen has an important drawback—the limited period of activity before resistance develops (7,20). Further, prolonged treatment with tamoxifen is associated with an increased risk for endometrial cancer due to significant agonist activity of the drug in uterus. As long as the ER is present in tumors, growth may still be stimulated by small amounts of estrogens or antiestrogens or by ligand-independent actions. The introduction of AI's for postmenopausal patients, either initially, or sequentially after tamoxifen, may produce better outcomes than the standard treatment of 5 years of tamoxifen (22-24). Nonetheless, in patients with advanced disease, only about ⅓ of HR+ tumors respond to AI's as first-line treatments (6). Further, resistance to AI's also develops due, in part, to E2-independent mechanisms (6,12,13). Consequently, a search has begun to find new antiestrogens that do not display agonist activity or lead to development of resistance. The first prototype drug, fulvestrant (25), is a pure ER antagonist that also exhibits a unique mechanism of action—downregulation of ER due in part to induced hyperubiquitination of ER (26,27). As fulvestrant has no agonistic activity but instead destabilizes ER, the drug elicits marked disruption of ER-mediated growth. However, fulvestrant has a major drawback—very low bioavailability—which is problemsome in the clinic. Although fulvestrant has activity in treating ER+ metastatic breast cancer in postmenopausal women with disease progression after tamoxifen or AI therapy (7,28,29), discovery of new ER antagonists with improved bioavailability and antitumor activity remains an important goal.
ER degradation limits hormone action. Ligand-induced down-regulation of ER is a pivotal step in halting E2 stimulation of growth, and the ubiquitin-proteasome pathway is the major system for selective degradation of such regulatory proteins (30). ERα was among the first of the nuclear receptors identified as substrates for this pathway (31-34). A common feature of proteasome-mediated protein degradation is covalent attachment of ubiquitin to lysine residues of proteins targeted for degradation followed by formation of polyubiquitin chains attached covalently to the protein. Ubiquitinated ERα is recognized and degraded by the multisubunit protease complex, the 26S proteasome (35). ER is degraded in a hormone-dependent manner, with this process contributing to regulation of hormone action; and the proteasome inhibitor, MG132, is well known to promote in vivo accumulation of ER and to block ligand-induced ER degradation (33). As noted above, the proteasome pathway also plays a critical role in interaction of ER with antagonists, such as SERMs and fulvestrant (27,34).
Therefore it would be useful to test new novel ER antagonists to find compounds which inhibit the growth of hyperproliferative cells, including breast cancer. Disclosed herein, inter alia, are solutions to these and other problems in the art.